Table 1.
List of primers used in the studya.
Figure 1.
Interaction between PCBP1 protein and the 5′UTR of EV71 RNA.
(A) Co-immunoprecipitation (Co-IP) and RT-PCR assay. RD cells were infected with EV71 at an MOI of 40 for 6 h and then cell extracts were prepared. For Co-IP assay, extract of EV71-infected cells was incubated with mouse anti-PCBP1 antibody (lanes 2 and 6), normal mouse IgG (lanes 3 and 7), or without antibody (-Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes 1–4) or for EV71 5′UTR RNA (lanes 5–8). Total RNA from cell extracts without Co-IP was evaluated by RT-PCR as a control (lanes 1 and 5). RT-PCR products were separated and detected by agarose gel electrophoresis and the expected band is indicated by an arrow. (B) Electrophoretic mobility shift assay (EMSA). Plasmid carrying EV71 5′UTR DNA was linearized and in vitro transcribed into RNA, which was then labeled with biotin-16-UTP at the 3′ end. Labeled RNA (0.5 µg) was incubated with recombinant PCBP1 (rPCBP1) at 0, 0.1, 0.15 and 0.2 µg (lanes 2 to 5) or BSA at 0.1, 0.15, and 0.2 µg (lanes 6 to 8), respectively. The RNA-protein complex (indicated by an arrow) was separated from free probe RNA and examined by EMSA. No-labeled RNA was in lane 1 as a control. (C) The specificity of the PCBP1 antibody. Purified rPCBP1 protein (lanes 1 to 4) and rPCBP2 protein (lanes 5 to 8) were subjected to SDS-PAGE at 0, 2, 4 and 8 µg, respectively, stained by Coomasie blue and then detected by specific PCBP1 antibody. (D) Biotinylated RNA/protein pull-down assays. For RNA/protein pull-down assay and for competition assay, cell extracts were mixed with biotinylated EV71 5′UTR RNA (lanes 2–9) along with different concentrations of nonbiotinylated yeast tRNA (lanes 2–5) or nonbiotinylated EV71 5′UTR RNA (lanes 6–9), respectively. After pull-down assay, the bound proteins were boiled and subjected to 12% SDS-PAGE and analyzed by western blotting with a mouse anti-PCBP1 antibody. The input contained 200 µg cell lysate (lane 1). (E) Extracts of RD cells (lanes 1–5), HeLa cells (lanes 6–10), and SK-N-SH cells (lanes 11–15) were prepared and then incubated without RNA (lanes 2, 7, and 12), with biotin-16-UTP (lanes 3, 8, and 13), non-biotinylated EV71 5′UTR (lanes 4, 9, and 14), or biotinylated EV71 5′UTR (lanes 5, 10, and 15). After pull-down assay, the bound proteins were boiled, eluted, and subjected to 12% SDS-PAGE and PCBP1 protein was detected by western blot with a mouse anti-PCBP1 antibody. The inputs were cell extracts of RD (lane 1), HeLa (lane 6), and SK-N-SH (lane 11).
Figure 2.
Determination of the sequences in 5′UTR of EV71 RNA required for the binding of PCBP1.
(A) The secondary structure of the full-length 5′UTR of EV71 RNA was predicted using the M-FOLD software (http://mfold.rna.albany.edu). In this predicted secondary structure, six domains (or stem-loops) were shown with first and last nucleotides numbered: domain I (nt 1 to 87), II (nt 111 to 179), III (nt 182 to 231), IV (nt 239 to 442), V (nt 450 to 559), and VI (nt 571 to 651). The three functional regions, cloverleaf, IRES, and linker, were underlined based on previous report [11]. (B) A series of plasmids carrying different deletions in the six stem-loops of EV71 5′UTR were generated: pcDNA3.0-5′UTR (T1, 1–100), (T2, 1–180), (T3, 100–230), (T4, 1–230), (T5, 100–450), (T6, 230–450), (T7, 1–450), and (T8, 1–565). (C) The plasmids were linearized and transcribed into RNA in vitro, which were then labeled with biotin-16-UTP. For the pull-down assay, cell extracts were mixed with these biotinylated EV71 5′UTR RNAs or nonbiotinylated RNAs as control (lanes 1, 3, 5, 7, 9, 11, 13, and 15). The complex was dissolved and PCBP1 protein was determined by western blot using a mouse anti-PCBP1 antibody (lanes 2, 4, 6, 8, 10, 12, 14, and 16). (D) EMSA assay for domain I (CL-I) and IV (SL-IV). Labeled RNA (CL-1 or SL-IV) (0.5 µg) was incubated with rPCBP1 at 0, 0.1, and 0.2 µg (lanes 2 to 4) or BSA at 0.2 µg (lane 5), respectively. No-labeled RNA was used as a control (lane 1). The RNA-protein complex was separated from free probe RNA and examined by an EMSA. The bound RNA is indicated by an arrow.
Figure 3.
Analysis of the function domain of PCBP1 protein required for its binding to the 5′UTR of EV71 RNA.
(A) The KH domains of full-length PCBP1 protein were predicted by PROSITE tools (http://prosite.expasy.org/). The amino acids of the three KH domains, KH1, KH2, and KH3, were underlined. (B) Based on the sequences of PCBP1 protein and its predicted domains, five plasmids expressing the full-length PCBP1 or truncated PCBP1 proteins were constructed, in which the KH3 was deleted (KH1-2), the KH1 was deleted (KH2-3), both KH2 and KH3 were deleted (KH1), or both KH1 and KH2 were deleted (KH3), respectively. (C) For the RNA-protein binding assay, HeLa cells were transfected with plasmids coding the Flag-tagged full-length PCBP1 (lanes 1-3) or the Flag-tagged truncated proteins, PCBP1-KH1-2 (lanes 4–6), PCBP1-KH2-3 (lanes 7–9), PCBP1-KH1 (lanes 10–12), and PCBP1-KH3 (lanes 13–15), respectively. For the pull-down assay, cell extracts were prepared and mixed with nonbiotinylated EV71 5′UTR RNA (lanes 2, 5, 8, 11, and 14) or biotinylated EV71 5′UTR RNA (lanes 3, 6, 9, 12, and 15), respectively. The complexes were boiled and dissolved and PCBP1 protein was determined by western blot analysis using anti-Flag antibody (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, and 15). The inputs were cell extracts without pull-down assay (lanes 1, 4, 7, 10, and 13).
Figure 4.
Subcellular distribution of PCBP1 protein during EV71 infection.
(A) RD cells were mock-infected or infected with EV71 at an MOI of 5 for 6 and 12 h. Cells were probed with rabbit anti-PCBP1 antibody and mouse anti-dsRNA antibody, stained with DAPI, examined by confocal microscopy (Fluoview FV1000; Olympus). PCBP1 (red), dsRNA (green), DAPI (blue). Bar = 20 µm. (B) RD cells were mock-infected or infected with EV71 at an MOI of 5 for 6 and 12 h. Cells were probed with mouse anti-PCBP1 antibody and rabbit anti-EV71 3C antibody, stained with DAPI and examined by confocal microscopy. PCBP1 (red), EV71 3C (green), DAPI (blue). Bar = 10 µm. (C) RD cells were infected with or without EV71 at an MOI of 5 for 12 h. Whole cell lysates, cytoplasm, and nucleus extractions were prepared from mock-infected and EV71-infected cells, respectively. Endogenous proteins were detected by western blot analysis using antibody to PCBP1 or β-actin. The blot is a representative of three independent experiments with similar results. The bar graph showed change of PCBP1 distribution based on three independent experiments with similar results (**, p<0.01).
Figure 5.
Localization of PCBP1 in ER-derived membrane-associated complex during EV71 replication.
(A) RD cells were mock-infected or infected with EV71 at an MOI of 5 for 8 h. Cells were probed with rabbit anti-PCBP1 antibody and mouse anti-dsRNA antibody, stained with ER-Tracker, observed by confocal microscopy. PCBP1 (red), dsRNA (green), ER-Tracker (blue). Bar = 20 µm. (B) Membrane flotation analysis of location of PCBP1. RD cells were infected with or without EV71 at an MOI of 5 for 8 h. Cell lysates from mock-infected and infected cells treated with or without Triton X-100 were separated by sucrose gradient sedimentation. Each fraction was blotted with specific antibodies to the ER marker protein (calnexin, CNX), PCBP1, and EV71 2C, respectively. Fractions 1 to 3 represent the membrane fraction. Fractions 4 to 9 represent the cytosolic fraction.
Figure 6.
Analysis of PCBP1 in the regulation of EV71 viral protein expression and virus production.
(A) RD cells were transfected with pCMV-PCBP1 (to over-express PCBP1), pCMV (a negative control of pCMV-PCBP1), siPCBP1 (to knock-down PCBP1) and siCtrl (a negative control of siPCBP1), respectively. At 24, 36, and 48 h post-transfection, cells extracts were prepared and proteins were detected by western blot using mouse anti-PCBP1 antibody or mouse anti-β-actin antibody. (B) RD cells were transfected with pCMV-PCBP1, pCMV, siPCBP1, and siCrtl, respectively. At 24, 36, and 48 h post-transfection, viability of transfected cells were determined by MTT assay. The viability of mock cells was normalized to 100%. Data are from three independent experiments with similar results. (C) RD cells were infected with or without EV71 at an MOI of 5 for 4, 8, and 12 h. Mock-infected and EV71-infected cells were lysed and proteins were detected by western blot analysis using antibodies to EV71 VP1 protein or β-actin protein. (D) RD cells were transfected with pCMV-PCBP1 at different concentrations (0, 1, 2, and 4 µg) or with pCMV at different concentrations (4, 3, 2, and 0 µg), siPCBP1 at different concentrations (0, 2.5, 5, 10 nM) or with siCtrl at different concentrations (10, 7.5, 5, 0 nM), respectively, for 36 h, and then infected with or without EV71 at an MOI of 5 for 12 h. Mock-infected and EV71-infected cells were lysed and proteins were detected by western blot analysis using antibodies to EV71 VP1 protein, PCBP1 or β-actin protein. (E) RD cells were transfected with pCMV-PCBP1, pCMV, siPCBP1, and siCtrl, respectively, for 36 h, and then infected with EV71 at an MOI of 5 for 4, 8, and 12 h. EV71-infected cells were lysed and proteins were detected by western blot analysis using antibodies to EV71 VP1 protein, PCBP1 or β-actin protein. (F) Cell culture supernatants from (D) were prepared and subjected to plaque assays and viral infectivity titers of culture supernatants (plaque forming units per ml, PFU/ml) on EV71-infected RD cells were determined. (G) Cell culture supernatants from (E) were collected and viral infectivity titers in supernatants were determined. Viral infectivity titers are expressed as means ± S.E. (n = 3) of three independent experiments (**, p<0.01; *, p<0.05).